Erk5 regulates MM apoptosis. (A) Expression of the dominant-negative form of Erk5 sensitizes MM cells to drug-induced apoptosis. Control (□) or HA-Erk5^AEF–expressing (▪) MM1S cells were plated at identical densities and treated for 24 hours with the indicated concentrations of PS341 or dexamethasone. MTT uptake was carried out as described in the text. The results represent the mean ± SD of quadruplicates of an experiment repeated twice. (B) Effect of PS341 on myeloma cells in the presence of bone marrow stromal cells (BMSC). Control or HA-Erk5^AEF–expressing MM1S cells were plated on wells previously coated, or not, with stromal cells. PS341 (at the indicated concentrations) was added together with the myeloma cells, and the experiment continued for 48 hours. BrdU was added for the last 8 hours. BrdU uptake was measured as described in “Materials and methods.” □ and ▪ indicate control and HA-Erk5^AEF–expressing MM1S cells with BMSC, respectively; ▦ and ▧, the same cells without BMSC, respectively. The results represent the mean ± SD of quadruplicates. (C) Action of PS341 on the levels of Erk5. The cell lines shown were treated with 10 nM PS341 for the indicated times, and then Erk5 or Erk1/2 levels were analyzed by Western blotting. (D) Effect of different caspase inhibitors on PS341-induced Erk5 down-regulation. MM1S cells were treated with PS341 in the presence or absence of the different caspase inhibitors (Z-VAD-FMK, 100 μM; Z-IETD-FMK and Z-LEHD-FMK, 50 μM) for 8 hours, and then Erk5 expression was analyzed by Western blotting. An aliquot of the cell extract (30 μg) was used to analyze caspase-3 cleavage by Western blotting. DMSO represents dimethylsulfoxide; Z-VAD-FMK represents Z-Val-Ala-DL-Asp fluoromethylketone; Z-IETD-FMK represents Z-Ile-Glu-Thr-Asp fluoromethylketone; Z-LEHD-FMK represents Z-Leu-Glu-His-Asp fluoromethylketone. (E) Effect of Erk5 overexpression on PS341-induced cell death. Expression of wild-type HA-Erk5 was carried out by transfection of pCDNA3 or pCDNA3-HA-Erk5 into MM1S cells, followed by G418 selection. A pool of transfected MM1S cells was selected, and the expression of exogenous HA-Erk5 was detected by Western blotting with anti-HA or with anti-Erk5 (top). The bottom panel shows the effect of PS341 on control or HA-Erk5–overexpressing MM1S cells. Cells were treated for 24 hours with 10 nM PS341, and the amount of apoptotic cells analyzed by annexin V–FITC staining. Data are represented as the mean ± SD of duplicates of an experiment repeated twice.